Hepatitis C virus (HCV) has a positive-sense single-strand RNA genome and is a member of the genus Hepacivirus within the Flaviviridae family of viruses (Rice, C. M. 1996 Flaviviridae: The viruses and their replication, In Fields Virology (B. N. Fields, D. M. Knipe, P. M. Howley, et al., Eds.), Third ed., pp. 931-959. Lippincott-Raven Publishers, Philadelphia). As for all positive-stranded RNA viruses, the genome of HCV functions as mRNA from which all viral proteins necessary for propagation are translated.
The viral genome of HCV is approximately 9600 nucleotides (nts) in length and consists of a highly conserved 5′ untranslated region (UTR), a single long open reading frame (ORF) of approximately 9,000 nts and a complex 3′ UTR. The 5′ UTR contains an internal ribosomal entry site (Tsukiyama-Kohara, K. et al. 1992 J. Virol. 66:1476-1483; Honda, M., et al. 1996 RNA 2:955-968). The 3′ UTR consists of a short variable region, a polypyrimidine tract of variable length and, at the 3′ end, a highly conserved region of approximately 100 nucleotides (Kolykhalov, A. A. et al. 1996 J. Virol. 70:3363-3371; Tanaka, T et al. 1995 Biochem. Biophys. Res. Commun. 215:744-749; Tanaka, T. et al. 1996 J. Virol. 70:3307-3312; Yamada, N. et al. 1996 Virology 223:255-261). The last 46 nucleotides of this conserved region were predicted to form a stable stem-loop structure thought to be critical for viral replication (Blight, K. J. and Rice, C. M. 1997 J. Virol. 71:7345-7352; Ito, T. and Lai, M. M. C. 1997 J. Virol. 71:8698-8706; Tsuchihara, K. et al. 1997 J. Virol. 71:6720-6726). The ORF encodes a large polypeptide precursor that is cleaved into at least 10 proteins by host and viral proteinases (Rice, C. M. 1996 Flaviviridae: The viruses and their replication, In Fields Virology (B. N. Fields, D. M. Knipe, P. M. Howley, et al., Eds.), Third ed., pp. 931-959. Lippincott-Raven Publishers, Philadelphia). The predicted envelope proteins contain several conserved N-linked glycosylation sites and cysteine residues (Okamoto, H. et al. 1992 Virology 188:331-341). The NS3 gene encodes a serine protease and an RNA helicase and the NS5B gene encodes an RNA-dependent RNA polymerase.
A remarkable characteristic of HCV is its genetic heterogeneity, which is manifested throughout the genome (Bukh, J. et al. 1995 Semin. Liver Dis. 15:41-63). The most heterogeneous regions of the genome are found in the envelope genes, in particular the hypervariable region 1 (HVR1) at the N-terminus of E2 (Hijikata, M. et al. 1991 Biochem. Biophys. Res. Commun. 175:220-228; Weiner, A. J. et al. 1991 Virology 180:842-848). HCV circulates as a quasispecies of closely related genomes in an infected individual. Globally, six major HCV genotypes (genotypes 1-6) and multiple subtypes (a, b, c, etc.) have been identified (Bukh, J. et al. 1993 PNAS USA 90:8234-8238; Simmonds, P. et al. 1993 J. Gen. Virol. 74:2391-2399).
The nucleotide and deduced amino acid sequences among isolates within a quasispecies generally differ by <2%, whereas those between isolates of different genotypes vary by as much as 35%. Genotypes 1, 2 and 3 are found worldwide and constitute more than 90% of the HCV infections in North and South America, Europe, Russia, China, Japan and Australia (Foms, X. and Bukh, J. 1998 Viral Hepatit. is Reviews 4:1-19). Throughout these regions genotype 1 accounts for the majority of HCV infections but genotypes 2 and 3 each account for 5-15%.
At present, more than 80% of individuals infected with HCV become chronically infected and these chronically infected individuals have a relatively high risk of developing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (Hoofnagle, J. H. 1997 Hepatology 26:15S-20S). The current treatment for chronic hepatitis C involves administration of interferon (IFN) and ribavirin, which induces a sustained response in less than 50% of treated patients, and which has a poorer response in genotype 1 compared to genotypes 2 and 3 (Davis, G. L. et al. 1998 N. Engl. J. Med. 339:1493-1499; McHutchison, J. G. et al. 1998 N. Engl. J. Med. 339:1485-1492). Consequently, HCV is currently the most common cause of end stage liver failure and the reason for most liver transplants performed in the U.S. (Hoofnagle, J. H. 1997 Hepatology 26:15S-20S). As a result of the inability to develop a universally effective therapy against HCV infection, it is estimated that there are still more than 25,000 new infections yearly in the U.S. (Alter, M. J. 1997 Hepatology 26:62S-65S).
Despite the intense interest in the development of vaccines and therapies for HCV, progress has been hindered by the absence of a useful cell culture system and the lack of any small animal model for laboratory study. For example, while replication of HCV in several cell lines has been reported, such observations have turned out not to be highly reproducible. In addition, the chimpanzee is the only animal model, other than man, for this disease. Consequently, HCV has been studied only by using clinical materials obtained from patients or experimentally infected chimpanzees, an animal model whose availability is very limited.
Kolykhalov, A. A. et al. (1997 Science 277:570-574) and Yanagi et al. (1997 PNAS USA 94:8738-8743 and 1998 Virology 244:161-172) reported the derivation from HCV strains H77 (genotype 1a) and HC-J4 (genotype 1b) of cDNA clones of HCV that are infectious for chimpanzees. However, while these infectious clones will aid in studying HCV replication and pathogenesis and will provide an important tool for development of in vitro replication and propagation systems, it is important to have infectious clones of multiple strains and genotypes, given the extensive genetic heterogeneity of HCV and the potential impact of such heterogeneity on the development of effective therapies and vaccines for HCV. The need for more tools to better characterize HCV and the need for more HCV therapeutics and diagnostic approaches is manifest.